One of the most significant developments in the field of bacterial pathogenesis research in the last few years has been the discovery that many bacterial pathogens have evolved complex specialized machines to transfer multiple bacterially-encoded proteins into eukaryotic cells. One of this type of machines is the type III secretion system (T3SS). A great deal of attention has been devoted to the study of these systems because they are essential virulence determinants for the bacterial pathogens that encode them. Proteins delivered by these machines have the capacity to modulate a variety of cellular functions and are collectively known as "effectors". Through work supported by this Grant, we have been studying a T3SS from Salmonella enterica serovar Typhimurium (S. Typhimurium), encoded within its pathogenicty island 1 (SPI-1). This system mediates several phenotypes that are essential for virulence including bacterial entry into and survival within non-phagocytic cells, the induction of programmed cell death in macrophages, and the stimulation of innate immune responses and inflammation in the intestinal tract. During the previous funding period we have made important discoveries that have significantly advanced our knowledge of this system and T3SSs in general. During the next funding period, we intend to continue this line of investigation, focusing our efforts on less-well understood aspects of T3SSs. More specifically, we propose to use a multidisciplinary approach to: 1) To investigate the function of the "export apparatus" of the S. Typhimurium SPI-1 T3SS;2) To investigate the function of InvA, a core component of the T3SS;3) To investigate the composition and assembly of the SpaO- organized platform;and 4) To obtain a high resolution in situ view of the S. Typhimurium SPI-1 T3SS. It is hoped that accomplishing these objectives will not only enhance our understanding of Salmonella spp. pathogenesis but also our understanding of T3SSs in general. Since this system is central to the pathogenesis of many important pathogenic bacteria, these studies may provide the bases for the development of broadly applicable anti-infective strategies. PUBLIC HEALTH RELEVANCE: Many important bacterial pathogens have evolved the capacity to "inject" proteins using a specialized nano-machine known as the type III secretion system. Bacterial proteins injected by this nanomachine manipulate the cells of the body for the pathogen's benefit. This Grant intends to better understand how this machine works in the bacterial pathogen Salmonella Typhimurium, which causes inflammatory diarrhea and is one of the most common causes of food poisoning. Since this system is central to the virulence of many important pathogenic bacteria, it is hoped that the understanding of the function of type III secretion systems will provide the bases for the development of broadly applicable anti infective drugs.